36] Assay of eukaryotic initiation factor eIF-2 activity with extracts of interferon-treated cells

Abstract

Publisher Summary This chapter presents that incubation of interferon-treated cell extracts with dsRNA leads to a reduction in their capacity to initiate protein synthesis. Thus, the incorporation of [ 35 S]methionine in the NH 2 -terminal positions of peptides synthesized in vitro is analyzed by a modification of the Edman degradation procedure and found to be reduced by dsRNA. Furthermore, the incorporation of [ 35 S]methionine into a peak sedimenting at approximately 40 S is measured by sedimentation on sucrose density gradients and also shown to be sensitive to dsRNA. The parallel activation of an endonuclease activity by dsRNA should not affect, the measurement of initiation complex formation. Since other steps in the protein synthetic pathway could be rate limiting, and it is attempted to compare the relative rates of initiation by direct measurement of the production of NH2-terminal methionine residues and have utilized both preinitiation complex formation and the Edman degradation procedure to give an overall view of the initiation process. The Edman procedure, however, has certain drawbacks. Activation of endonuclease activity causes degradation of mRNA, which probably results in fewer intact 5' termini being available for initiation; this probably explains the limited ability of initiation factors to reverse the inhibition.